Development of Reverse Transcription-PCR Assays Specific for Detection of Equine Encephalitis Viruses

Virus strains.

Each of the alphaviruses used in this study represented one of the currently classified alphavirus species (21). A description of the isolates, including the year and location of isolation, as well as their passage history, was published earlier (25). To evaluate each RT-PCR assay using a panel of virus strains, nine additional virus isolates each of EEE and WEE were chosen from the virus collection of the Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control, Fort Collins, Colo. The selection of isolates was intended to include those with maximal geographical distribution (South and North America) and various dates of virus isolation (Table 1). To further determine the specificity or cross-reactivity of the RT-PCR, representative strains of the 12 VEE subtypes were used as described earlier (14, 25). Recent VEE isolates from Mexico and Peru were used as the original sera or their first cell culture passage (Table 2).

Extraction of viral RNA.

Viral RNA was extracted from a 200-μl aliquot of infectious Vero cell culture supernatant according to the method of Lewis et al. (18). The viral RNA was rehydrated in 50 μl of diethyl pyrocarbonate (DEPC)-treated water (25). The amount of RNA of each preparation was measured photometrically (GeneQuant; Pharmacia), and the number of RNA molecules was calculated on the basis of a genome length of 11,700 nucleotides and an average weight for each nucleotide of 336.3 g/mol, yielding a weight of 6.53 × 10⁻⁹ ng per RNA molecule.

Primer selection.

The oligonucleotide primers were chosen from the aligned nucleotide sequences of the structural polyprotein coding regions of Sindbis (38), Ockelbo (35), Aura (31), Ross River (8), O'Nyong Nyong (17), Chikungunya (M. Parker, GenBank accession no. L37661), Semliki Forest (10), EEE (7, 46, 47, 50), WEE (12), and representative strains of the VEE subtype viruses (14). For each virus-specific detection, two primer pairs each for primary RT-PCR and subsequent nested PCR were synthesized. Their sequences and genomic locations are presented in Table 3 and Fig. 1.

General PCR procedures.

All RT-PCRs and nested PCRs were carried out in 0.2 ml of thin-walled PCR tubes using a model 9600 or a model 2400 thermocycler (Perkin-Elmer Corp.). The final reaction volume was always 100 μl. During optimization of each of the specific RT-PCR and nested-PCR assays, the amount of the following reagents was varied: primers (0.1 to 1 μM, in 0.1 μM steps), MgCl₂ (1 to 4 mM, in 0.5 mM steps), and deoxynucleoside triphosphate (dNTP) (100, 150, and 200 μM; MBI Fermentas). The remaining reagents were identical in all RT-PCRs (5 mM dithiothreitol, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.01% gelatin, 2.5 U of TaqExtender PCR additive (Stratagene, Heidelberg, Germany), 2 U of RAV-2 reverse transcriptase (Amersham, Braunschweig, Germany), 2 U of AmpliTaq DNA polymerase (Perkin-Elmer Corp., Weiterstadt, Germany), 5 μl of RNA template, and DEPC-treated water to give a final volume of 100-μl). Nested PCRs were performed in 100-μl reactions containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.01% gelatin, 5 mM dithiothreitol, 1.5 U of AmpliTaq DNA polymerase, and 2 μl of cDNA template from the previous RT-PCR. The annealing temperatures tested in both RT-PCRs and nested PCRs ranged from 55 to 72°C. For both RT-PCRs and nested-PCRs, the optimal concentrations of MgCl₂ and dNTP were consistently 2 mM and 200 μM, respectively. Optimal results were defined as the virus-specific amplification obtained with the primer pairs, concentrations of reaction components, and cycling conditions that allowed the most sensitive detection of the respective viral RNA. When differing reactions resulted in the same sensitivity, criteria such as the lack of signs of nonspecific background bands were used as additional parameters to measure the success of the optimization.

To prevent carryover contaminations, pipetting of the reaction mixtures for the RT-PCRs was performed under a hood in a room separated from both the room where amplicons were analyzed by gel electrophoresis and the room where the pipetting of the nested PCRs was done. A 10-μl aliquot of each reaction was loaded onto 2% agarose gels (Eurogentec, Heidelberg, Germany) in TBE buffer (0.1 M Tris-HCl, pH 8.0; 0.091 M boric acid; 2 mM disodium EDTA). DNA fragments were visualized after staining with ethidium bromide on a UV transilluminator at 302 nm and recorded using a charge-coupled device CCD camera (Fröbel, Wasserburg, Germany) and the FotoTouch Color software (Logitech).

All RT-PCR and nested-PCR were tested with genomic RNA template of representative strains of all known alphavirus species to ensure virus specificity within the genus.

EEE-specific RT-PCR and nested PCR.

Cycling conditions of the uninterrupted RT-PCR program were 30 min at 50°C followed by one cycle with denaturing at 94°C for 90 s, annealing at 64°C for 90 s, and extension at 72°C for 90 s and 35 cycles of 94°C for 20 s, 64°C for 30 s, and 72°C for 20 s. The final extension step was prolonged to 5 min to ensure completion of the length of the amplicon. Optimal results were obtained with 0.1 μM concentrations each of primers EEE-4 and cEEE-7 (Table 3, combination II).

A 2-μl aliquot of the RT-PCR mixture was subjected to a nested PCR which, after an initial denaturation at 94°C for 90 s, consisted of 25 cycles of 94°C for 20 s, 65°C for 35 s, and 72°C for 17 s. The final elongation step was 4 min. EEE-5 and cEEE-6 primer concentrations of 0.3 μM each (combination IIn) yielded the best results.

WEE-specific RT-PCR and seminested PCR.

For the specific amplification of WEE, a set of three primers targeting the E2 gene and implemented as RT-PCR and seminested PCR (Table 3, combinations I and Isn) were found to be 300 times more sensitive than a RT-PCR–nested PCR primer set (combinations II and IIn) hybridizing to the E2 and E1 genes embracing the 6K gene (Fig. 1; see also below). The reaction conditions were as follows: 30 min at 50°C; one cycle consisting of 90 s at 94°C, 60 s at 68°C, and 90 s at 72°C; 35 cycles consisting of 20 s at 94°C, 30 s at 68°C, and 17 s at 72°C; and a final extension step of 5 min for the RT-PCR I (Table 3). Each primer (WEE-1 and cWEE-3) was at 0.2 μM, while in the subsequent seminested PCR (combination Isn) 0.3 μM concentrations each of primers WEE-2 and cWEE-3 gave the best results. The cycling conditions were as described for the EEE-specific nested PCR, except that the annealing temperature was set at 63°C and the elongation time was only 15 s.

VEE-specific RT-PCR and seminested PCR.

To develop VEE-specific primers, the aligned 26S RNA sequence data of representative strains of the 12 VEE subtype varieties were used (14) and compared with the available sequence data of other alphaviruses (see above). The level of nucleotide identity among VEE subtype varieties IAB, IC, and ID was >94%, while this level dropped to 70 to 88% when VEE-IAB was compared to the other subtype varieties (14). To avoid the use of degenerate primers, we focused on the genetically closely related and medically important VEE-IAB, -IC, -ID, -II, and -IE subtype varieties. Unfortunately, highly homologous regions among them also displayed a high level of nucleotide identity to EEE or WEE in these regions. Hence, two forward and three reverse primers were designed to function in RT-PCR–nested-PCR combinations (Table 3, Fig. 1).

After we tested the specificity and sensitivity of each combination, we found that performing the RT-PCR with primers VEE-2 versus cVEE-4 (Table 3, combination IV) with a seminested PCR using primers VEE-2 versus cVEE-3 (combination IIIsn) yielded the best results (see below). The optimal reaction conditions were found to be an RT for 30 min at 50°C, followed by one cycle of 90 s at 94°C, 90 s at 61°C, and 90 s at 72°C and 35 cycles, each consisting of 20 s at 94°C, 40 s at 61°C, and 17 s at 72°C. The final extension step was 5 min. For this RT-PCR (IV), 0.2 μM concentrations of primers VEE-2 and cVEE-4 gave the best results. In the subsequent seminested PCR (IIIsn), the cycling conditions were as described for the WEE-specific seminested PCR (Isn), with VEE-2 and cVEE-3 primer concentrations of 0.3 μM each found to give the best results.

Investigation of serum samples.

The VEE-specific RT-PCR assay was applied to human sera gathered as part of the “febrile illness surveillance in the Amazon River Basin.” From all of the serum samples listed in Table 2 virus was isolated in either Vero cells, C6/36 cells, or newborn mice and subsequently identified as VEE in an indirect immunofluorescence assay as described previously (44). A Mayaro virus-positive serum sample served as a negative control. RNA extracted from this serum was amplified by using a genus-specific RT-PCR–seminested PCR as described earlier (25). Compared to the VEE-specific RT-PCR–seminested PCR method described above, the following slight modifications were made: the viral RNA was extracted with the QIAmp viral RNA kit according to the supplier's recommendation (Qiagen), no TaqExtender PCR additive was used, SuperScript reverse transcriptase (2 U; Gibco BRL) was used instead of RAV-2 reverse transcriptase, and the annealing temperature was set at 55°C in the model 9600 thermocycler for both RT-PCR and seminested PCR.

EEE-specific RT-PCR and nested PCR.

Based on the nucleotide and the deduced amino acid sequence alignments of the available alphaviral 26S RNA sequence data, a total of 10 regions with virus-specific nucleotide sequences sufficiently distinct from the remaining alphaviruses were identified. Primers for three regions showed a high degree of self-complementarity and were not used. Of the remaining seven regions, eight primers (one region was used for both a forward and a reverse primer) were made, resulting in two sets of RT-PCR–nested-PCR primer pairs. With the first RT-PCR (I in Table 3, and Fig. 1) Aura virus yielded an amplicon of about 600 bp, which was slightly smaller than the EEE-specific RT-PCR product of 635 bp (not shown). In the subsequent nested PCR (In), the 10 tested EEE strains exclusively yielded the expected 284-bp fragment rendering the I and In combinations EEE specific. The sensitivity of this combination was 3 × 10⁵ RNA molecules in the RT-PCR and 3 × 10³ RNA molecules in the nested PCR (not shown). The second set of primers for RT-PCR (II; EEE-4 versus cEEE-7) yielded the expected 464-bp product with all of the EEE strains tested (Fig. 2A) and did not amplify the RNA of any other alphavirus (not shown). Weak bands of 100 bp were visible for most alphaviruses and the negative control, suggesting them to be of primer dimer origin. In the respective nested PCR (IIn), the 262-bp fragment was also only amplified with the EEE strains (Fig. 2B). None of the other alphaviruses showed a visible amplicon (not shown). The second combinations (II plus IIn) were 100-fold more sensitive than the previous combination, detecting 3 × 10³ (RT-PCR) and 30 RNA molecules (nested PCR), respectively (Fig. 2C).

WEE-specific RT-PCR and seminested PCR.

Eleven regions with sufficient sequence divergence compared to the other alphaviruses were identified for WEE. Four possible primer sequences were excluded because of predicted primer dimer formations. The remaining seven sites were used to design primers for use in a RT-PCR–seminested PCR (Table 3, combinations I and Isn) and a RT-PCR–nested PCR (combinations II and IIn) (Fig. 1). Testing of the specificity of the RT-PCR primers revealed the expected 354-bp amplicon for all 10 WEE and slightly smaller amplicons for Mucambo, Pixuna, and Babanki viruses for RT-PCR I (Fig. 3A). The second primer pair (II) amplified only the 10 WEE by RT-PCR (not shown). In the subsequently performed seminested or nested round of amplification, either combination (Isn or IIn) amplified the expected band (195 or 506 bp, respectively) specifically for the 10 WEE strains tested (shown for Isn in Fig. 3B). Although the first RT-PCR (I) was only WEE specific in conjunction with the seminested PCR (Isn), it was found to be 300 times more sensitive than the WEE-specific combinations II plus IIn. The detection limits were 6 × 10⁶ and 6 × 10³ RNA molecules for combinations II plus IIn, respectively. The RT-PCR of combination I detected 2 × 10³ RNA molecules. This sensitivity was increased by a factor of 100 in the seminested PCR (Isn), allowing the detection of as few as 20 RNA molecules (Fig. 4).

VEE-specific RT-PCR and seminested PCR.

All five primer combinations (Fig. 1) exclusively amplified VEE and no other alphavirus when used in RT-PCRs (not shown). However, their reactivity with the various VEE subtypes differed considerably. While all five RT-PCRs amplified VEE-IAB, -IC, -ID, and -II equally well, RNA of VEE-IE strain Mena II was amplified by RT-PCRs I and II to a much lower extent. None of the five RT-PCRs amplified the RNA of VEE-IF strain 78V-3531. RT-PCR I was the only RT-PCR that amplified the remaining VEE subtypes. RT-PCR II additionally amplified the three varieties of VEE subtype III (IIIA, IIIB, and IIIC), while RT-PCR III additionally amplified RNA of VEE-IIIB, -IIIC, and -IV. Only RT-PCRs IV and V reacted specifically with the subtype viruses (VEE-IAB, -IC, -ID, -IE, and -II) that the primers had been designed for (shown for RT-PCR IV in Fig. 5A). Smaller bands of lighter intensity of about 100, 250, and 300 bp were produced with the other VEE subtypes tested (Fig. 5A). Because detection of VEE-IE is important in light of the recent outbreaks in Mexico, RT-PCRs I and II were not chosen for further testing. For a subsequent round of amplification, three seminested PCRs using the templates from RT-PCR IV or V were possible, i.e., IIIsn (194 bp) out of IV or V and IVsn (342 bp) out of V. Cross-reactivity among the VEE subtypes existed with the first combinations (IV and IIIsn) for VEE-IIIB, -IIIC, and -IV (Fig. 5B), which resembled the reaction pattern of RT-PCR III. In the combinations V plus IIIsn, the 194-bp band was additionally produced for VEE subtypes IIIB and IV, while the second combinations (V plus IVsn) did not amplify RNA of VEE subtypes other than VEE-IAB to IE, and II (data not shown). We determined the sensitivity of combinations IV plus IIIsn and V plus IIIsn for both VEE-IAB strain Trinidad donkey (TRD) and for VEE-IE strain Mena II. The sensitivity for VEE-IAB was the same for both RT-PCR and seminested-PCR combinations, allowing the amplification of 20 RNA molecules of strain TRD (shown for IV plus IIIsn in Fig. 5C). For the amplification of VEE-IE strain Mena II, combinations IV plus IIIsn detected 70 RNA molecules of VEE subtype variety IE. This was 10 times more sensitive than the V and IIIsn combinations with a detection limit of 700 RNA molecules.

VEE-specific combinations IV plus IIIsn were successfully applied to serum samples known to be VEE positive by virus isolation. By using RT-PCR IV, one serum sample (IQT 8131) showed the specific 342-bp amplicon. All samples that were once passaged in cell cultures or suckling mouse brain displayed the specific 342-bp band as well (Fig. 6A). After the subsequent seminested PCR (IIIsn), all samples, including the serum samples that had been negative after RT-PCR, showed the VEE-specific 194-bp amplicon (Fig. 6B).